The overall aim of this research proposal is to apply functional methodology (initially developed at UW and adapted to high throughput by Regulome Corporation) for quantitative chromatin profiling to delineate the functional DNA elements across 30 megabases of human genome sequence selected by the ENCODE working group (the 'ENCODE regions'). First we will produce a quantitative chromatin profile of the ENCODE regions in a human lymphoblastoid cell line. This profile will enable precise localization of all major Dnasel-hypersensitive sites ('HSs' hereafter). Quantification of sequence-specific Dnasel sensitivity will be accomplished through application of a novel, tested high-throughput quantitative real-time PCR assay ('Hypersensitivity qPCR' or 'HSqPCR'). A specialized primer set that spans the ENCODE regions at high resolution will be designed and synthesized. This primer set will be employed in all the experiments. Second, we will produce quantitative chromatin profiles of the ENCODE regions in 3 additional cell types derived from the 3 major embryonic lineages (endoderm, mesoderm, ectoderm). These profiles will enable localization of all major HSs present in each tissue type. The profiles, in combination with that of lymphoid cells, will further enable identification of tissue or lineage-restricted HSs and also of sites present in multiple lineages. Third, we will perform confirmatory validation experiments on identified HSs using high-resolution hypersensitivity Southerns. Hypersensitivity Southerns represent an extensively applied 'gold-standard' methodology for identification of HSs. 250 sites per year will be selected from those identified with the high throughput method and validated with this independent methodology. Fourth, the quantitative chromatin profiles produced will be subjected to detailed computational analysis with the aim of determining the correspondence between HSs and evolutionarily-conserved non-coding sequences. Parallel analyses will focus on identification of DNA sequence motifs associated with tissue-specific, lineage-specific, or multilineage HSs. Algorithms for identification and classification of HSs in the human genome will be developed and tested. The performance of selected published algorithms for identification of active regulatory sequences will also be evaluated. It is expected that this research effort will result in the identification of the majority (if not all) of the regulatory elements of the ENCODE regions, and will provide information on the feasibility of the application of this functional approach to the identification of all regulatory elements of the human genome.